Lipid enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Filoviruses are lipid-enveloped viruses that have a filamentous lipid-envelope and despite being discovered more than 40 years ago, not much is known on how they acquire their lipid coat. These viruses bud from the plasma membrane of the host cell and cause viral hemorrhagic fever with up to a 90% fatality rate. Filoviruses include Ebola virus (EBOV) and Marburg virus (MARV), which are classified as category A pathogens by the NIH as they pose a serious public health and national security risk. These viruses harbor a negative sense RNA genome that encodes seven genes. The viral matrix protein VP40, which regulates budding from the host cell membrane, underlies the viral lipid envelope. VP40 is a peripheral protein and the only protein required from these viruses to form filamentous virus like particles that are nearly indistinguishable from authentic virions. Since little is known about how VP40 interacts with biological membranes, many fundamental questions about Filovirus assembly and budding remain unanswered. Preliminary studies demonstrate that a host cell enzyme, phospholipase D, is required for sufficient viral particle release and VP40 particle displacement at the plasma membrane. Phospholipase D is an enzyme that uses phosphatidylcholine as a substrate to generate the anionic lipid phosphatidic acid (PA). Preliminary studies also demonstrate that VP40 protein expression in human cells is sufficient to increase cellular levels of PA. The central hypothesis of this proposal is that the host enzyme phospholipase D (PLD) plays an essential role in the late stage budding of filoviruses. This R21 application describes experiments to provide a cohesive cellular and biochemical model of the role of PLD activity and PA generation in Filovirus budding. Specific Aim 1 will investigate the role of PLD activity and PA generation in the cellular assembly and budding of EBOV and MARV. We will elucidate the potential for inhibition of synthesis of this glycerophospholipid to inhibit Filovirus budding. Experiments with a BSL-4 collaborator will also test this hypothesis against authentic EBOV and MARV. Specific aim 2 will investigate the mechanism by which PA stabilizes VP40 oligomers and the molecular origin of PA binding by VP40. Quantitative measurements in our live cell BSL-2 surrogate system will determine the kinetics of VLP assembly and release and the role of PLD activity and PA generation in stabilizing VP40 oligomers at the plasma membrane for the scission process. The molecular origins of PA binding will be investigated using strong rationale of lipid-protein interactions and the known VP40 structures. Taken together, these studies should produce new and important mechanistic insight into how Filovirus particles form from the plasma membrane of cells.